Regenerative potential is inextricably tied to youthfulness and declines with aging, prompting research into mechanisms of rejuvenation to enhance myocardial repair, particularly in the middle aged to elderly patients suffering from acute myocardial injury or chronic heart failure. Regenerative potential and cellular pluripotency is linked to regulation of the Lin28 / let-7 signaling axis, which is also associated with myocardial protection from pathologic injury. Lin28 is employed in this proposal to define a novel heterogeneous cardiac interstitial cell (CIC) population possessing youthful characteristics and enhanced functional competency. The Lin28 interstitial population (LIP) defies categorization as a specific cell type, consisting of a combinatorial mix used as a foundation to select for naturally occurring CICs with youthful phenotype. This proposal advances fundamental innovative principles of CIC and cardiac stem cell (CSC) biology by revealing a new conceptual approach to the problem of identifying, propagating, and deploying the most biologically active cells to effect reparative and regenerative processes. Implementation is facilitated by our expertise in working with fresh and cultured CICs from both murine and human sources coupled with our legacy of over a decade engaged in seminal CSC research. The long term goal of this proposal is to deliver novel insight regarding CIC biology that can be modified, enhanced, and altered to promote myocardial healing. The short term goal is to contextualize LIPs and their inherent Lin28 / let-7 signaling axis relative to CIC and CSC in pursuit of mitigating myocardial damage and enhancing reparative processes. Specific Aims are: 1) LIPs are a cardiac interstitial cell population with distinct but heterogeneous phenotypic and functional signature 2) Lin28 expression and LIPs change in response to pathological injury as well as aging, 3) Derivation of LIP cultures resulting from in vitro expansion amplifies a subpopulation with phenotypic characteristics influenced by passage number and co- culture environment, and 4) Functional adaptation of LIPs depends upon and is regulated by the Lin28 / let-7 signaling axis. The innovation of this proposal is discovery and advancement of novel CIC biology through identification, characterization, and manipulation of a heterogeneous cell population coupled with a canonical regeneration-associated signaling axis to potentiate myocardial reparative processes. The significance of these studies is implementation of a marker that defines a novel CIC subset defined by pluripotent ?youthful? characteristics rather than traditional cell type-specific markers, advancing use of a combinatorial cell treatment approach defined by inherent population reparative potential rather than compartmentalized functional activity of any single cell category. These studies provide fundamental knowledge to generate novel hypotheses and approaches to enhance myocardial response to pathologic injury.